Method And Dyes For Detecting And Destroying Cancer Cells

ABSTRACT

This invention relates to new carbocyanine dye compositions, pharmaceutical compositions comprising such compositions, methods of detecting via near infrared fluorescent imaging incipient cancer cells and selective destruction of cancer cells identified by administration of such pharmaceutical compositions. A method of detecting and destroying cancer cells includes introducing a gold dye into an organism suspected of having a cancer cell. The gold dye is a carbocyanine dye covalently attached to a gold nanoparticle. A near infrared light is shined on a region suspected of having the cancer cell. Fluorescence from the gold dye is detected. A beam of radio frequency energy is directed at the region to induce hyperthermia in the cancer cell. The carbocyanine dye has the most basic structure of MHI-148 and structures 6 and 22 with a Au n —[S—CH 2 (CH 2 ) 9 CH 2 —(OCH 2 CH 2 ) 4 O]COCH 2 CH 2 -phenyl-O group on a cyclohexene ring that imparts activity to the cancer cell binding and destruction processes.

RELATED APPLICATIONS

This United States patent application is a continuation of U.S. patentapplication Ser. No. 12/778,569, filed May 12, 2010, now U.S. patentSer. No. 10/030,036, issued Jul. 24, 2018, which claims the benefit ofU.S. Provisional Patent Application No. 61/178,835, filed May 15, 2009,each hereby incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

The present invention was co-developed by CTI and KPS Technologies, LLC.

REFERENCE TO A SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTING

Not Applicable

BACKGROUND OF THE INVENTION

Traditionally cancer has been treated with general radiation treatmentand chemotherapy. Both of which have been shown to have a number of sideeffects. For instance, radiation treatment can result in loss of hair,nausea, and low white cell counts and other side effects such asabdominal pain, nausea, and vomiting. As a result, there has been aconstant desire to find cancer treatments with fewer side effects. Onesolution has been to create RF (Radio Frequency) absorption enhancersthat are attached to antibodies that attach to cancer cells. These RFabsorption enhancers are very small tuned circuits that inducehyperthermia in the cancer cells to which they are attached when RFenergy is directed at them. Unfortunately, these RF absorption enhancersdo not provide feedback on where the cancer cells are located. As aresult, the RF energy cannot be highly directed to the area with thecancer cells. In addition, these RF absorption enhancers are complex tomanufacture.

Thus, there exists a need for an improved method of treating cancer thatdoes not have the side effects of chemotherapy or high intensityradiation treatment and in which the energy used to kill the cancercells can be highly targeted.

BRIEF SUMMARY OF INVENTION

This invention relates to new carbocyanine dye compositions,pharmaceutical compositions comprising such compositions, methods ofdetecting via near infrared fluorescent imaging incipient cancer cellsand selective destruction of cancer cells identified by administrationof such pharmaceutical compositions.

A method of detecting and destroying cancer cells includes introducing agold dye into an organism suspected of having a cancer cell. The golddye is a carbocyanine dye covalently attached to a gold nanoparticle. Anear infrared excitation light source is scanned on a region suspectedof having the cancer cells. Fluorescence emitted from the excited golddye is detected by near infrared imaging techniques. A beam of radiofrequency energy is directed at the near infrared fluorescence imagedregion to induce hyperthermia in the cancer cell. In one embodiment, thecarbocyanine dye has the most basic structure of MHI-148 with aAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group on acyclohexene ring that imparts activity to the cancer cell binding anddestruction processes. Note that multiple gold atoms may be attached tothe phenyl-O group. In another embodiment, silver, copper or anothermetal may be substituted for gold.

These gold dyes are easy to produce and comparatively inexpensive. Thegold dye is rapidly eliminated from the healthy cells and tissue eithervia urinary and fecal routes with no accumulation in the healthy tissue.Certain gold dye structures with optimized total charge on the moleculehave been discovered that avoid passage through the liver during cleanout from the body. Any side effects of the process are thereforeminimized.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a flow chart of the steps used in a method for detecting anddestroying cancer cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to new carbocyanine dye compositions,pharmaceutical compositions comprising such compositions, methods ofdetecting via near infrared fluorescent imaging incipient cancer cellsand selective destruction of cancer cells identified by administrationof such pharmaceutical compositions.

FIG. 1 is a flow chart of the steps used in a method for detecting anddestroying cancer cells. The process starts, step 10, by introducing agold dye into an organism suspected of having cancer at step 12. Thegold dye is a carbocyanine dye covalently attached to a metalnanoparticle. In a preferred embodiment, the metal nanoparticle is gold.Near infrared light is shone on a region of the organism suspected ofhaving cancer cells at step 14. Fluorescence from the gold dye isdetected at step 16. The gold dye adheres to cancer cells and any excessof the gold dye is quickly flushed from organism's body. As a result,fluorescence only occurs in areas that have cancer cells. At step 18, abeam of radio frequency energy is directed at the region to inducehyperthermia in the cancer cell, which ends the process at step 20.

The gold dye is flushed from the body along with the dead cancer cells.A number of mechanismS have been hypothesized as to why the gold dyeinduces hyperthermia and this invention is not limited to any specifictheory. It is known that metal particles less than 100 micrometers areheated by microwave energy. One theory is that the metal nanoparticlesare directly heated by the microwave energy. It is possible that themagnetic field of the radio frequency energy induces inductive heatingof the metal nanoparticles. The optimum frequency of the radio waves canbe determined by measuring the heat output versus the frequency. Theheat output can be measured using an infrared thermal camera.

The carbocyanine dye of the gold dye is known to absorb light in thenear infrared spectrum from 680 to 1200 nm and to fluoresce in the nearinfrared spectrum at longer wavelengths. These carbocyanine dyes havebeen shown to selectively bind to cancer cells and cancerous tissue.

Specific structures were designed that selectively bind to cancer cellsand cancerous tissue in-vitro and in-vivo after covalent attachment ofone or more carbocyanine dye molecule to gold nanoparticles. Goldparticles that do not have dyes attached to their surface are separatedand not used. A specific dye composition that facilitates the transportof gold particles synthesized according to Scheme 1 selectively binds tocancer cells in-vivo and accumulate in tumors. Any non-binding gold dyecleans out rapidly without toxic effects. The example dye shown herecontains bromide counter anions but in manufactured pharmaceuticalcompositions the bromide will be changed to a pharmaceuticallyacceptable carrier or excipient such as citrate, tartrate, etc. Suchcarriers or excipients refer to an excipient that can be included in thecompositions of the invention and that causes no significant adversetoxicological effects to the patient.

Note that the final structure of scheme 2 is structure 6 and the finalstructure of scheme 3 is structure 22.

The preferred formula 1-8 of the carbocyanine dyes for the presentinvention have a Au_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-Ogroup on the cyclohexene or cyclopentene ring of the heptacyaninestructures disclosed that may have a significant role in the describedretaining and toxicity properties. In the preferred embodiment,heptamethine cyanine dyes are synthesized. The preferred heptamethinecyanines absorb light efficiently and have maximum absorptionwavelengths in the region of 780 to 1100 nm. This wavelength is suitablefor reducing background fluorescence in vivo and corresponds to theradiation wavelength of the GaAlAs diode laser and other semiconductorlight sources.

Some of these compounds in formula 1-8 and the compounds shown in Scheme1 have variable water solubility, near infrared fluorescent spectralproperties suitable for in-vivo tissue imaging (e.g. MHI-148 has λ_(max)absorption 780 nm and high intensity λ_(max) emission 800 nm (FIG. 1)),and have been tested to have similar activities in near infraredfluorescent imaging of tumor cells and inhibiting tumor cell growth invitro, and permit selective destruction of any gold labeled cancer cellsvia radiofrequency treatment. The solubility of the labeled goldparticles may be controlled by the dye molecules attached to the surfaceof gold particles or by additional solubilization groups attached to thegold particle surface as it is described by the literature or by priorart. Biodistribution of these gold particles are determined by themolecules attached to the gold particle surface and particle size.

Formula 1 shows cyanine dyes having the most basic structure of MHI-148with a Au_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group on acyclohexene ring that imparts activity to the cancer cell binding anddestruction processes. In this embodiment, when X isAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group, the maximumabsorbance wavelength is between 770 and 780 nm, depending on thesolvent and the maximum emission wavelength (maximum quantum yield andlambda fluorescent light) is between 805 and 820 depending on thesolvent.

Formula 2 shows cyanine dyes having an extended conjugation in theheterocyclic moiety and containing cyclopentene ring. In thisembodiment, when X is aAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group, the maximumabsorbance wavelength is between 790 and 800 nm, depending on thesolvent and the maximum emission wavelength (maximum quantum yield andlambda fluorescent light) is between 810 and 820 nm depending on thesolvent. This extended conjugation slightly increases the absorption andemission wavelengths and at the same time increases the hydrophobicityof the molecule that seems to be an important factor in dye retention bythe cancer cells.

Formula 3 shows cyanine dyes having a five member ring in the center ofthe molecule. In this embodiment, when X is with aAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group, the maximumabsorbance wavelength is between 770 and 790 nm, depending on thesolvent and the maximum emission wavelength (maximum quantum yield andlambda fluorescent light) is between 790 and 810 nm, depending on thesolvent. The five member ring may slightly increase the reactivity ofthe halogen for reactions leading to Au dyes. Note, the kill mechanismin this invention is RF energy and localized heating of cancer cellsnear to the Au dye.

Formula 4 shows cyanine dyes having an extended conjugation in theheterocyclic moiety and containing the five member central ring. In thisembodiment, when X isAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group, the maximumabsorbance wavelength is between 770 and 790 nm, depending on thesolvent and the maximum emission wavelength (maximum quantum yield andlambda fluorescent light) is between 790 and 810 nm depending on thesolvent. This extended conjugation slightly increases the absorption andemission wavelengths and at the same time increases the hydrophobicityof the molecule that seems to be an important factor in dye retention bythe cancer cells.

Formula 5 shows cyanine dyes having an extended conjugation in theheterocyclic moiety and containing the six member central ring. In thisembodiment, when X isAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group, the maximumabsorbance wavelength is between 770 and 790 nm, depending on thesolvent and the maximum emission wavelength (maximum quantum yield andlambda fluorescent light) is between 790 and 810 nm depending on thesolvent. This extended conjugation slightly increases the absorption andemission wavelengths and at the same time increases the hydrophobicityof the molecule that seems to be an important factor in dye retention bythe cancer cells.

Formula 6 shows cyanine dyes having an extended conjugation in theheterocyclic moiety and containing the five member central ring. In thisembodiment, when X isAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group, the maximumabsorbance wavelength is between 770 and 810 nm, depending on thesolvent and the maximum emission wavelength (maximum quantum yield andlambda fluorescent light) is between 790 and 810 nm depending on thesolvent. This extended conjugation slightly increases the absorption andemission wavelengths and at the same time increases the hydrophobicityof the molecule that seems to be an important factor in dye retention bythe cancer cells.

Formula 7 shows synthesized cyanine dyes having an extended conjugationin the heterocyclic moiety and containing the six member central ring.In this embodiment, when X isAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group, the maximumabsorbance wavelength is between 980 and 1000 nm, depending on thesolvent and the maximum emission wavelength (maximum quantum yield andlambda fluorescent light) is between 1000 and 1020 nm depending on thesolvent. This extended conjugation substantially increases theabsorption and emission wavelengths and at the same time increases thehydrophobicity of the molecule that seems to be an important factor indye retention by the cancer cells.

Formula 8 shows synthesized cyanine dyes having an extended conjugationin the heterocyclic moiety and containing the five member central ring.In this embodiment, when X isAu_(n)—[S—CH₂(CH₂)₉CH₂—(OCH₂CH₂)₄O]COCH₂CH₂-phenyl-O group, the maximumabsorbance wavelength is between 980 and 1000 nm, depending on thesolvent and the maximum emission wavelength (maximum quantum yield andlambda fluorescent light) is between 1000 and 1020 nm depending on thesolvent. This extended conjugation substantially increases theabsorption and emission wavelengths and at the same time increases thehydrophobicity of the molecule that seems to be an important factor indye retention by the cancer cells.

Other suitable dyes include but not limited to unsymmetricaltricarbocyanine, pentacyanine and heptacyanine structures functionalizedwith gold nanoparticles 3.5-5 nm (TEM) as indicated in Formula 9 whichabsorb light having the wavelengths in the region of 680 to 760 nm.

Formulas 1-9 show a variety of carbocyanines dyes attached to a metalnanoparticle (gold dye) that selectively attach to cancer cell. Thecarbocyanine dye fluoresces in the near infrared range and can beactivated by light in the near infrared spectrum. These gold dyes arequickly flushed from the body except for those molecules that attach tocancer cells. When the gold dye is attached to a cancer cell orcancerous tissue and a RF field is applied the cancers materialundergoes hyperthermia and is destroyed. Very little of the surroundinghealthy tissue is affected.

Thus there has been described an improved method of treating cancer thatdoes not have the side effects of chemotherapy or radiation treatmentand in which the energy used to kill the cancer cells can be highlytargeted.

While the invention has been described in conjunction with specificembodiments thereof, it is evident that many alterations, modifications,and variations will be apparent to those skilled in the art in light ofthe foregoing description. Accordingly, it is intended to embrace allsuch alterations, modifications, and variations in the appended claims.

What is claimed is:
 1. A method of detecting and destroying cancercells, comprising the steps of: introducing a gold dye into an organismsuspected of having a cancer cell, wherein the gold dye is acarbocyanine dye covalently attached to a metal nanoparticle; shinning anear infrared light on a region suspected of having the cancer cell;detecting a fluorescence from the gold dye; targeting a beam of radiofrequency energy at the region to induce hyperthermia in the cancercell.
 2. The method of claim 1, further including the step of notinducing hyperthermia in non-cancer cells near the cancer cell.
 3. Themethod of claim 1, wherein the step of introducing the gold dye includesattaching the metal nanoparticle by anAu_(n)—[S—CH2(CH2)9CH2-(OCH2CH2)4O]COCH2CH2-phenyl-O group on acyclohexene ring of the carbocyanine dye.
 4. The method of claim 3,further including synthesizing the carbocyanine dye represented byFormula
 1. 5. The method of claim 4, wherein the gold nanoparticle isattached to a cyclohexene ring of the carbocyanine dye.
 6. The method ofclaim 1, wherein the step of shinning the near infrared light includesthe step of selecting a GaAlAs diode laser with a predeterminedwavelength.
 7. A heptamethine cyanine dye covalently attached to a metalparticle having a maximum absorption wavelength in the region between780 and 1100 nm.
 8. The heptamethine cyanine dye of claim 7, wherein theheptamethine cyanine dye is represented by Formula
 1. 9. Theheptamethine cyanine dye of claim 7, wherein the heptamethine cyaninedye is represented by Formula
 2. 10. The heptamethine cyanine dye ofclaim 7, wherein the heptamethine cyanine dye is represented by Formula3.
 11. The heptamethine cyanine dye of claim 7, wherein the heptamethinecyanine dye is represented by Formula
 4. 12. The heptamethine cyaninedye of claim 7, wherein the heptamethine cyanine dye is represented byFormula
 5. 13. The heptamethine cyanine dye of claim 7, wherein theheptamethine cyanine dye is represented by Formula
 6. 14. Theheptamethine cyanine dye of claim 7, wherein the heptamethine cyaninedye is represented by Formula
 7. 15. The heptamethine cyanine dye ofclaim 7, wherein the heptamethine cyanine dye is represented by Formula8.
 16. The heptamethine cyanine dye of claim 7, wherein the bromidecounter anions are replaced with an pharmaceutically acceptable carrier.17. The heptamethine cyanine dye of claim 7, wherein the bromide counteranions are replaced with an pharmaceutically acceptable excipient. 18.An unsymmetrical cyanine dye with a covalently attached to a metalparticle as represented in Formula 9 having a maximum absorptionwavelength in the region between 680 to 760 nm.
 19. The unsymmetricalcyanine dye of claim 18, wherein the unsymmetrical cyanine dye is atricarbocyanine.
 20. The unsymmetrical cyanine dye of claim 18, whereinthe unsymmetrical cyanine dye is a pentacarbocyanine.
 21. Theunsymmetrical cyanine dye of claim 18, wherein the unsymmetrical cyaninedye is a heptacarbocyanine.
 22. A symmetrical cyanine dye of thestructure
 6. 23. A symmetrical cyanine dye of the structure
 22. 24. Asymmetrical cyanine dye of structure 6 attached to a functionalized goldnanoparticle AuS—[CH2]9-(OCH2CH2)4OH via an ester bond.
 25. Asymmetrical cyanine dye of structure 6 attached to a functionalized goldnanoparticle Au—S—[CH2]9NH2 via an amide bond.
 26. A symmetrical cyaninedye of structure 6 attached to a functionalized gold nanoparticleAu—S—[CH2]9-SH via a thioester bond
 27. A symmetrical cyanine dye ofstructure 22 attached to a functionalized gold nanoparticleAuS—[CH2]9-(OCH₂CH₂)4OH via an ester bond.
 27. A symmetrical cyanine dyeof structure 22 attached to a functionalized gold nanoparticleAu—S—[CH2]9NH2 via an amide bond.
 28. A symmetrical cyanine dye offormula 22 attached to a functionalized gold nanoparticle Au—S—[CH2]9-SHvia a thioester bond.
 29. A symmetrical cyanine dye of structure 6 or 22in which the O-phenyl-CH2CH2CO2H group is replaced by S[CH2]9S—Au_(n).30. A method of detecting and destroying cancer cells, comprising:introducing a composition into an organism suspected of having cancercells; wherein said composition comprises a carbocyanine dye covalentlyattached to a metal nanoparticle via a linker bound to a cyclohexenering or a cyclopentene ring of said carbocyanine dye; wherein saidlinker comprises a polyethylene group; and wherein said compositionselectively binds to said cancer cells; shining a near infrared light ona region suspected of having said cancer cells; detecting a fluorescencefrom said composition to locate said cancer cells; and targeting a beamof radio frequency energy at said region to induce hyperthermia in saidcancer cells.
 31. The method of claim 30, wherein said fluorescence isemitted by said carbocyanine dye.
 32. The method of claim 30, whereinsaid composition does not bind to healthy cells.
 33. The method of claim30, wherein said carbocyanine dye absorbs light having a wavelengthranging from between about 680 nanometers to about 1200 nanometers. 34.The method of claim 30, wherein said carbocyanine dye comprises aheptamethine cyanine dye.
 35. The method of claim 34, wherein saidheptamethine cyanine dye absorbs light having a wavelength ranging frombetween about 780 nanometers to about 1100 nanometers.
 36. The method ofclaim 30, wherein said induction of hyperthermia is facilitated by saidmetal nanoparticle.
 37. The method of claim 30, wherein said metalnanoparticle comprises a gold nanoparticle.
 38. The method of claim 30,wherein said metal nanoparticle has a diameter of less than about 100micrometers.
 39. The method of claim 30, wherein said linker comprises asulfur.
 40. The method of claim 30, wherein said carbocyanine dye has astructure selected from the group consisting of: tricarbocyanine,pentacarbocyanine and heptacarbocyanine.
 41. A method of detecting anddestroying cancer cells, comprising: introducing a composition into anorganism suspected of having cancer cells; wherein said compositioncomprises a carbocyanine dye covalently attached to a metal nanoparticlevia a linker bound to a nitrogen of an indole moiety of saidcarbocyanine dye; wherein said linker comprises a polyethylene group;and wherein said composition selectively binds to said cancer cells;shining a near infrared light on a region suspected of having saidcancer cells; detecting a fluorescence from said composition to locatesaid cancer cells; and targeting a beam of radio frequency energy atsaid region to induce hyperthermia in said cancer cells.
 42. The methodof claim 41, wherein said fluorescence is emitted by said carbocyaninedye.
 43. The method of claim 41, wherein said composition does not bindto healthy cells.
 44. The method of claim 41, wherein said carbocyaninedye absorbs light having a wavelength ranging from between about 680nanometers to about 1200 nanometers.
 45. The method of claim 41, whereinsaid carbocyanine dye comprises a heptamethine cyanine dye.
 46. Themethod of claim 45, wherein said heptamethine cyanine dye absorbs lighthaving a wavelength ranging from between about 780 nanometers to about1100 nanometers.
 47. The method of claim 41, wherein said induction ofhyperthermia is facilitated by said metal nanoparticle.
 48. The methodof claim 41, wherein said metal nanoparticle comprises a goldnanoparticle.
 49. The method of claim 41, wherein said metalnanoparticle has a diameter of less than about 100 micrometers.
 50. Themethod of claim 41, wherein said linker comprises a sulfur.
 51. Themethod of claim 41, wherein said carbocyanine dye has a structureselected from the group consisting of: tricarbocyanine,pentacarbocyanine and heptacarbocyanine.